When developing antibody pharmaceuticals with a drug action mechanism based on antibody-dependent cell-mediated cytotoxicity (ADCC), it is important to select clones with high ADCC activity. ADCC activity is evaluated using cells expressing an antigen of interest (target cells) and effector cells that kill those target cells. Effector cells recognize the Fc region of antibodies bound to the target cells via the Fcγ receptor (FcγR). Signals transmitted from FcγR causes the effector cells to kill the target cells. FcγR binds to a molecule called the γ chain through its transmembrane domain, and transmits ADCC signals via this γ chain (Non-patent Documents 1 to 3). Mouse FcγR3 and FcγR4, and human FcγR3 are known as FcγR4 that induce ADCC. Amino acid sequence comparisons of the transmembrane domains of human and mouse FcγR4 show that five out of the 21 amino acids are different between human FcγR3 and mouse FcγR3, and seven out of the 21 amino acids are different between human FcγR3 and mouse FcγR4. Human γ chain and mouse γ chain comparisons show that one out of the 21 amino acids is different between the sequences in the transmembrane domains (Non-patent Document 4).
When measuring the ADCC activity of human antibodies, human NK cells are used as effector cells. Human NK cells can be purified from human peripheral blood mononuclear cells (PBMC) using the NK Cell Isolation Kit II (Miltenyi Biotec K.K.). Alternatively, PBMC can be used directly as effector cells. PBMC can be purchased (from Cambrex Corporation), or can be prepared from fresh peripheral blood collected from volunteers. However, when such cells are used as effector cells, the drawbacks include lot-to-lot differences and laborious preparation.
To avoid such drawbacks, systems that use human NK cell lines as effector cells have been developed for measuring the ADCC activity of human antibodies. The NK92 human NK cell line (ATCC) does not express human FcγR, but expresses the human γ chain (Non-patent Document 5). Therefore, ADCC activity can be induced by forcedly-expressing human FcγR3 in the NK92 human NK cell line (Non-patent Documents 6 and 7). This greatly reduced preparation labor and enabled accurate measurements having small lot-to-lot differences. Furthermore, it has been reported that chimeric molecules produced by fusing the extracellular domain of human FcγR3 and the transmembrane domain and intracellular domain of human γ chain induce ADCC activity related to human antibodies (Non-patent Document 8).
On the other hand, when measuring the ADCC activity of mouse antibodies, mouse spleen cells are used as effector cells (Non-patent Documents 9 and 10). To prepare mouse spleen cells, it is necessary to remove the spleen from mice, hemolyze erythrocytes, and activate NK cells with interleukin 2. However, since spleen cells prepared in this manner have high natural killer activity to kill target cells in an antibody-independent manner, the ADCC activity may not be measurable depending on the type of target cells. Furthermore, preparation of the effector cells requires effort.
Systems for measuring the ADCC activity of human antibodies using human NK cell lines have been developed. However, since the use of mouse NK cell lines is generally unknown, a system for conveniently measuring the ADCC activity of mouse antibodies using an NK cell line has not been established. Furthermore, since there are sequence differences between Fcγ receptors and γ chains between human and mouse, even if mouse FcγR is expressed as it is in human NK92 cells, mouse FcγR will not be able to bind to the human γ chain with similar strength as human FcγR.
Therefore, to measure the ADCC activity of mouse antibodies, it was necessary to use a method that requires much effort such as the method of preparing mouse spleen cells as described above, or the method of preparing a chimeric antibody in which the antibody Fc regions have been replaced with those of a human antibody.
Prior art literature information relating to the present invention is shown below.    [Non-patent Document 1] Blood 2003, 101, 4479.    [Non-patent Document 2] J. Immunol. 1991, 146, 1571    [Non-patent Document 3] Immunol. Lett. 2004, 92, 199    [Non-patent Document 4] J. Biol. Chem. 2006, 281, 17108    [Non-patent Document 5] Leukemia Res. 2003, 27, 935    [Non-patent Document 6] 97th AACR annual meeting 2006, abstract number 635    [Non-patent Document 7] J. Biol. Chem. 2004, 279, 53907    [Non-patent Document 8] Blood 2006, 107, 4669    [Non-patent Document 9] Oncol. Rep. 2006, 15, 361    [Non-patent Document 10] Cell. Immunol. 1988, 115, 257